Yeast organisms produce a number of proteins which are synthesized intracellularly, but which have a function outside the cell. Such extracellular proteins are referred to as secreted proteins. These secreted proteins are expressed initially inside the cell in a precursor or a pre-protein form containing a pre-peptide sequence ensuring effective direction of the expressed product (into the secretory pathway of the cell) across the membrane of the endoplasmic reticulum (ER). The pre-sequence, normally named a signal peptide, is generally cleaved off from the desired product during translocation. Once entered in the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi the protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium (Pfeffer, S. R. and Rothman, J. E. Ann.Rev.Biochem. 56 (1987) 829-852).
Several approaches have been suggested for the expression and secretion in yeast of proteins heterologous to yeast. European published patent application No. 88 632 describes a process by which proteins heterologous to yeast are expressed, processed and secreted by transforming a yeast organism with an expression vehicle harbouring DNA encoding the desired protein and a signal peptide, preparing a culture of the transformed organism, growing the culture and recovering the protein from the culture medium. The signal peptide may be the signal peptide of the desired protein itself, a heterologous signal peptide or a hybrid of native and heterologous signal peptides.
A problem encountered with the use of signal peptides heterologous to yeast might be that the heterologous signal peptide does not ensure efficient translocation and/or cleavage ot the precursor polypeptide after the signal peptide.
The Saccharomyces cerevisiae MF.alpha.1 (.alpha.-factor) is synthesized as a pre-pro form of 165 amino acids comprising signal- or pre-peptide of 19 amino acids followed by a "leader" or pro-peptide of 64 amino acids, encompassing three N-linked glycosylation sites followed by (LysArg((Asp/Glu)Ala).sub.2-3.alpha.-factor).sub.4 (Kurjan, J. and Herskowitz, I. Cell 30 (1982) 933-943). The signal-leader part of the pre-pro MF.alpha.1 has been widely employed to obtain synthesis and secretion of heterologous proteins in S. cerevisiae.
Use of signal/leader peptides homologous to yeast is known from i. a. U.S. Pat. No. 4,546,082, European published patent applications Nos. 116 201, 123 294, 123 544, 163 529 and 123 289 and DK patent application No. 3614/83.
In EP 123 289 utilization of the S. cerevisiae .alpha.-factor precursor is described whereas WO 84/01153 indicates utilization of the S. cerevisiae invertase signal peptide and DK 3614/83 utilization of the S. cerevisiae PH05 signal peptide for secretion of foreign proteins.
U.S. Pat. No. 4,546,082, EP 16 201, 123 294, 123 544 and 163 529 describe processes by which the .alpha.-factor signal-leader from S. cerevisiae (MFa1 or MF.alpha.2) is utilized in the secretion process of expressed heterologous proteins in yeast. By fusing a DNA sequence encoding the S. cerevisiae MF.alpha.1 signal/leader sequence at the 5' end of the gene for the desired protein secretion and processing of the desired protein was demonstrated.
EP 206 783 discloses a system for the secretion of polypeptides from S. cerevisiae using an .alpha.-factor leader sequence which has been truncated to eliminate the four .alpha.-factor units present on the native leader sequence so as to leave the leader peptide itself fused to a heterologous polypeptide via the .alpha.-factor processing site LysArgGluAlaGluAla (SEQ ID NO:51). This construction is indicated to lead to an efficient processing of smaller peptides (less than 50 amino acids). For the secretion and processing of larger polypeptides, the native .alpha.-factor leader sequence has been truncated to leave one or two of the .alpha.-factor units between the leader peptide and the polypeptide.
A number of secreted proteins are routed so as to be exposed to a proteolytic processing system which can cleave the peptide bond at the carboxy end of two consecutive basic amino acids. This enzymatic activity is in S. cerevisiae encoded by the KEX 2 gene (Julius, D. A. et al., Cell 37 (1984b) 1075). Processing of the product by the KEX 2 protease is needed for the secretion of active S. cerevisiae mating factor .alpha.1 (MF.alpha.1 or .alpha.-factor) whereas KEX 2 is not involved in the secretion of active S. cerevisiae mating factor a.
Secretion and correct processing of a polypeptide intended to be secreted is obtained in some cases when culturing a yeast organism which is transformed with a vector constructed as indicated in the references given above. In many cases, however, the level of secretion is very low or there is no secretion, or the proteolytic processing may be incorrect or incomplete resulting in secretion of a considerable amount of leader bound product polypeptide. Prosequences, and especially N-terminally located prosequences, or leader sequences expressed in eucaryotic cells, such as yeast cells, are extensively glycosylated, cf. Fiedler and Simons, Cell, 81, p 309-312; and Moir, D. T., Yeast mutants with increased secretion efficiency, in Yeast Genetic Engineering, Barr, P. J., Brake, A. J., and Valenzuela, P. eds., wherein a general review of glycosylation and secretion of proteins is presented. It is generally recognised that glycosylation, which may be either N-linked, O-linked, or both, is important for efficient transport through the secretory pathway, cf. Caplan et al., Journal of Bacteriology, Vol. 173, No. 2, p. 627-635; and Jars et al., The Journal of Biological Chemistry, Vol. 270, No. 42, p 24810-24817. Moreover, due to the extensive glycosylation the purification of secreted propeptides is difficult and differs considerably from the processing steps that are typically employed for the purification of the mature secreted polypeptide. Clements et al., Gene, 106 (1991) 267-272, have shown that using a eucaryotic consensus signal sequence and two 19-aa pro-sequences comprising fractions of the .alpha.-Factor leader and identical except for the presence or absence of a potential Asn linked (N-linked) glycosylation site for secretion of hEGF from yeast had no effect on secretion, and the level of secretion was comparable to the level obtained when using the .alpha.-Factor prepro-sequence (about 3 .mu.g/ml).
Expression of heterologous proteins as fusion proteins is a well known concept and has been utilized in various contexts in different organisms. Secretory expression of a heterologous protein in yeast is often performed as a fusion protein with a secretion prepro-leader to confer secretion competence. Prepro-leaders tend to be hyperglycosylated or extensively O-linked glycosylated in the S. cerecisiae secretory pathway. Purification of hyperglycosylated fusion protein is laborious due to its heterogeneous nature. Efficient prepro-leaders lacking hyperglycosylation, with no or limited O-linked glycosylation and replacement of the dibasic Kex2 endoprotease site with a more convenient enzymatic processing site, provide an alternative to conventional yeast expression by purification of the fusion protein and subsequently in vitro maturation with a suitable enzyme as exemplified herein for the insulin precursor. In vitro maturation of a purified fusion protein is more flexible since dependency on the Kex2 endoprotease is eliminated and any proteolytic enzyme can be used for maturation provided that the heterologous protein does not have any internal processing sites. Purification of the fusion protein from the culture supernatant followed by in vitro maturation will avoid N-terminal processing of the heterologous protein by dipeptidyl aminopeptidase. Secretion of a fusion protein rather than the heterologous protein has the advantage that the propeptide may increase stability and solubility until purification and maturation. Secretory expression in yeast of heterologous proteins with internal dibasic sites may lead to Kex2 endoprotease processing and a decrease in fermentation yield. This can be avoided by utilizing a secretion prepro-leader lacking N-linked glycosylation to confer secretion competence, introduction of a suitable enzyme processing site between the prepro-leader and the heterologous protein, expression in a Kex2 endoprotease negative S. cerevisiae strain followed by purification and in vitro maturation.
It is an object of the present invention to provide novel synthetic leader peptides or pro-sequences which ensure a higher yield and a more efficient recovery and/or processing of polypeptides, preferably secreted polypeptides, including leader bound polypeptides, and polypeptides being fused N-terminally to peptide sequences including leader sequences and/or spacer sequences each of which optionally being separated from the other constituent sequences by a processing site, expressed in a eucaryotic host cell organism, preferably a fungal cell, such as a yeast cell or a filamentous fungus cell.